The proposed collaboration between the U.S. investigator (Dr. Peter Melby, UTHSCSA, San Antonio) and the foreign investigator (Dr. Sara Robledo, University of Antioquia, Medellin, Colombia) is an extension of NIH grant R01AI61624, on which Dr. Melby is the PI. Experimental animal studies have identified a dominant role for alternatively activated macrophages (AAMs) in the pathogenesis of cutaneous and visceral leishmaniasis, but their role in human susceptibility and disease has not been determined. The proposed FIRCA grant will extend the experimental animal studies to humans by investigating a previously studied population of individuals who were either classified as resistant (no clinical infection but positive DTH reaction to Leishmania antigen) or susceptible (chronic non-healing cutaneous leishmaniasis). Previous in vitro studies determined that macrophages from susceptible individuals had reduced capacity to control Leishmania infection compared to macrophages from resistant individuals, but the mechanism of this susceptibility is unknown. The overall hypothesis of this proposal is that nonhealing or progressive leishmaniasis is mediated through alternative macrophage activation, which impairs parasite killing. For all of the proposed studies, an in vitro model of Leishmania- infected monocyte-derived macrophages (MDMs), isolated from resistant or susceptible individuals, will be used. The first Specific Aim will test the hypothesis that macrophages from susceptible individuals respond directly to Leishmania infection through a program of alternative activation. The MDMs will be infected with L. (V.) panamensis (dermatropic strain) or L. (L.) donovani (viscerotropic strain), and markers of classical (NO production, NOS2 expression, and CCL3 production) and alternative macrophage activation (arginase activity, IL-10, CCL17, and CCL22 production, and expression of CD23) will be used to determine if Leishmania infection directly induces alternative activation, and if it is associated with susceptibility. The second Specific Aim will test the hypothesis that macrophages from susceptible individuals, when infected with Leishmania, become more sensitive to alternative activation effect of type 2 cytokines. Exposure of uninfected and Leishmania-infected MDMs to IL-4, IL-10, or IL-13 will determine if there is an additive or synergistic effect of the parasites and cytokines in the activation of STAT6 and upregulation of AAM genes. The third Specific Aim will use siRNA- mediated knockdown of STAT6 to test the hypothesis that the program of alternative macrophage activation is STAT6-dependent, and that inhibition of STAT6 activation in susceptible human macrophages will enhance their IFN-3-induced anti-leishmanial response. PUBLIC HEALTH RELEVANCE: Leishmaniasis is a vector borne disease that is endemic throughout much of the world, for which control strategies have largely been unsuccessful or non-sustainable. Cutaneous leishmaniasis is an emerging and re-emerging disease in many parts of the world, and in Colombia the number of cases of cutaneous leishmaniasis has increased dramatically in the past 4 years. Recent epidemics of visceral leishmaniasis have resulted in several hundred thousand deaths in India and Sudan. The poor response to chemotherapy and paucity of available drugs make investigation into the mechanisms of disease, and the identification of preventive or therapeutic interventions, of paramount importance.